Comparison of molecular methods for Bartonella henselae detection in blood donors

The Bartonella genus consists of neglected pathogens associated with potentially transfusional-transmitted and fatal human diseases. We aimed to evaluate Bartonella sp. prevalence in 500 blood donors and compare the results with the data already published about these samples. We used molecular diagnostic methods to detect Bartonella sp.-DNA from blood and liquid culture samples: (A) conventional PCR for two gene regions, the ITS targeting the genus Bartonella and the specific gltA Bartonella henselae; (B) nested PCR for the ftsZ gene and (C) qualitative real-time PCR for the gltA gene, both B. henselae specific. We obtained 30/500 (6%) DNA detections from the blood samples; 77/500 (15.4%) DNA detections from liquid culture samples and five (1%) samples had DNA detection from both. In total, we detected B. henselae DNA from 102/500 (20.4%) donors. The samples used in this study had already been submitted for Bartonella sp.-DNA detection using only a conventional PCR in liquid culture. Sixteen samples (3.2%) were positive previously, and from these 16 samples, 13 were negative in the new investigation. We concluded that the use of liquid culture combined with different molecular tests increases the possibility of detecting Bartonella sp.-DNA, but the tests do not avoid false-negative results. More than a fifth of blood donors had at least one PCR that detected Bartonella sp.-DNA among the eight molecular reactions performed now (four reactions in whole blood and four in liquid culture). Seven percent had B. henselae-DNA detection for two or more distinct regions. Considering the results obtained previously, the DNA of Bartonella spp. was detected or the agent isolated in 23% of analyzed blood donors. The results establish that the low bacteremia and the fastidious characteristics of the bacterium are challenges to laboratory diagnosis and can make it difficult to confirm the infection in patients with bartonelloses.

The genus Bartonella has more than 47 species and subspecies, and at least 17 of them have already been related to clinical manifestations in humans [5,7]. Of these, three species are associated with the largest number of diseases: Bartonella bacilliformis, Bartonella quintana and Bartonella henselae [11], the latter being the most frequent species [12][13][14].
Blood-sucking arthropods are the main mode of Bartonella transmission [6,24,25]. Other types of transmission, such as percutaneous accidents, transplantation of solid organs (kidney and liver), and vertical transmission, have been linked with infections by Bartonella spp. [22,[26][27][28][29]. Transmission by blood transfusion is also possible. In a case reported by Pons et al., transmission of B. bacilliformis was described after platelet transfusion from an asymptomatic blood donor [30]. A study conducted at UNICAMP showed that B. henselae remained viable for 35 days in experimentally infected blood bags stored at 4˚C [31]. This study was used by the American Association of Blood Banks (AABB) to include the bacterium as a pathogen that can be transmitted by blood transfusion. In another study, Ruiz et al. demonstrated that B. bacilliformis remained viable in samples collected from patients with symptoms of Carrión disease after 30 months of storage at 4˚C [32]. A study by Silva et al. detected the DNA of B. henselae in the spleen of mice that received transfusion of blood from animals that had been experimentally infected, although molecular blood tests were negative in all transfused animals [33]. Asymptomatic blood donors infected by Bartonella spp. may carry this bacterium in his red blood cells, posing a real risk of infection to blood recipients. However, laboratory confirmation of Bartonella sp. infection remains a major challenge. The fastidious nature of this genus, even in a specific culture medium, limits the diagnostic use of blood or tissue cultures [4,8,14,34]. Molecular methods have expanded the detection of Bartonella sp.; however, no current diagnostic method is able to confirm infection by Bartonella spp. in all infected patients, since these bacteria have low bacteremia, which makes detection even more difficult [ A published study investigated the prevalence of Bartonella species in a population of blood donors using just one conventional blood liquid culture PCR. Blood samples from 500 voluntary blood donors were incubated in BAPGM (Bartonella alpha-proteobacteria growth medium) liquid medium and cultured at 37˚C in 5% CO 2 for 14 days. Then, the samples were subinoculated on agar medium containing 30% sheep blood for another 42 days. DNA was extracted from the liquid culture and tested by Bartonella sp.-specific conventional PCR, which amplifies the ITS region. The amplified products were sequenced to identify the species. Gram-negative isolates obtained from solid culture were also tested by the same technique. Sixteen blood donors (3.2%) were positive for Bartonella spp. in PCR after culture in liquid and solid media. DNA sequencing confirmed the homology of 15 samples with B. henselae and one sample with Bartonella clarridgeiae [37,38].
The present study aimed to evaluate the presence of Bartonella-DNA in blood and liquid culture of 500 blood donor samples from the University of Campinas (UNICAMP) Blood Bank, Campinas, Sao Paulo, Brazil, using four different PCRs in whole blood and in liquid culture samples and compare the results obtained in the previous project and already published [37], since the samples were the same.

Ethics statement
This project was submitted to the University of Campinas Institutional Review Board (IRB) under n˚122/2005, and formal written consent was obtained from donors who agreed to participate in the research. In 2015, the IRB reapproved it under n˚1.135.941 for further tests.

Samples
This study analyzed two samples (whole blood and liquid culture) of each 500 blood donors from the University of Campinas (UNICAMP) Blood Bank, randomly collected from November 2009 to January 2010 during a blood donation procedure. These were the same samples used in a previous study that has already been published [37]. Epidemiological data about blood donors, such as gender; occupational animal exposure; contact with cats, dogs, other companion animals, bites from dogs, cats, and other animals; arthropod bites caused by ticks, fleas, or other insects; previous blood transfusion, etc., were already analyzed and published [39].
Whole blood samples collected in tubes with EDTA and liquid culture (another aliquot of whole blood incubated in BAPGM liquid medium and cultured at 37˚C in 5% CO 2 for 14 days) were stored at -20˚C.

DNA extraction
DNA extraction from whole blood and from liquid culture was performed using a QIAamp DNA Mini Kit (Qiagen). Controls were added to each extraction following the protocol already described [37].

PCR
Controls were used in each reaction, and molecular techniques were performed carefully to avoid contamination following the procedures described in previous work [37].
All samples (both DNA extracted from the whole blood and liquid culture) were tested for all PCR techniques described below. Promega enzyme (GoTaq Flexi) was used in all reactions, except for qualitative real-time PCR. The PCR primers and conditions are described in Table A in S1 Appendix.

Quality control PCR
The quality of the extracted DNA and the absence of PCR inhibitors in DNA samples were tested by the amplification of a fragment of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, which encodes a glycolysis enzyme expressed by all mammalian cells [40].

Conventional PCR
Two different reactions for the target gene were performed: the ITS region, or the 16S-23S rRNA intergenic region, for Bartonella spp.

Nested PCR
A species-specific nested PCR was used in this study for the target region that encodes protein FtsZ that plays a role in cell division of B. henselae [43].
All PCR products were submitted to electrophoresis in 1.5% agarose gel stained with GelRed and visualized in a photodocumenter with UV light.

Qualitative real-time PCR
The samples were tested by real-time PCR using the same primers used in conventional PCR for amplification of the citrate synthase gene (gltA) in the Sybr Green system using enzyme Fast SYBR Green Master Mix (ThermoFisher Scientific) [42]. In this study, real-time PCR results were used as qualitative PCR, considering results as positive or negative. In addition to the amplification curve, the melt curve was analyzed in comparison with the curve of the diluted Bartonella DNA used as a positive control. We considered positive samples with melt curves between 73.08˚C and 73.41˚C. We also performed electrophoresis, and the positive samples were confirmed by the presence of a band in the 1.5% agarose gel stained with GelRed.

Sequencing
Amplified DNA with enough concentration was sent for Sanger sequencing. The results were analyzed using Chromas 2.6.6 software and compared to the GenBank database using the BLAST tool from the National Center for Biotechnology Information (NCBI).

Statistical analysis
The McNemar-Bowker test was performed to decide which type of sample (blood or liquid culture) would be the most appropriate considering the results of any PCR method. This test is an extension of the McNemar test when there are nominal variables with more than two categories of nonindependent variables. Bayesian latent class model (LCM) analysis was performed to find the best species-specific PCR method for B. henselae, regardless of the tested sample, and the best PCR method for blood or liquid culture samples. This test assumes that none of the tests is perfectly accurate; then, an 'imperfect gold standard' model is defined according to the results of multiple diagnostic tests with the same samples. The Bayesian approach can infer the prevalence of the studied agent and the test properties by adjusting the possibility of conditional dependence between the tests [44]. The limiting factor of Bayesian LCM analysis refers to the need for intense computer programming. In our study, an online tool was used (Modeling of Infectious Disease Centre-Imperfect Gold Standard Model) with results from three different PCR methods analyzing the same samples. The number of repetitions of the analysis was set at 25,000. Accuracy measures such as sensitivity, specificity, positive predictive value, and negative predictive value [45] were calculated using this method, as well as the 95% confidence interval. All p values were considered statistically significant if p < 0.05.
Additionally, the receiver operating characteristic (ROC) curve method was applied. This analysis uses a simple graphic method to study the variation in sensitivity and specificity for different cutoff values. The area under the ROC curve (AUC) is associated with the discriminatory power of a diagnostic test [46].

PCR
All participants had whole blood and liquid culture analyzed, and all extracted samples had amplified in the quality control PCR (GAPDH), demonstrating the presence of DNA and the absence of PCR inhibitors.
For a better understanding, the results are presented in Venn diagrams, showing how many samples were amplified in each reaction and detected in more than one PCR (Fig 1). Fig 1A shows the results of the PCR tests performed with the DNA extracted from whole blood samples. Nested and real-time PCR were the most sensitive tests for this type of sample, and only one sample had simultaneous detection in all four tests. Qualitative real-time PCR was the most efficient test for DNA extracted from liquid culture, showing amplification of 60 samples. Only four samples had simultaneous detection in all four PCR tests ( Fig 1B). Fig 1C  shows the results of all PCR tests performed with the DNA extracted from blood and liquid culture samples. The most efficient reaction to detect Bartonella spp. was real-time PCR with amplification of 72 samples, 45 of which were only in this test. Note that Fig 1C is not a product of the sum of the two previous Venn graphs. An example is that a sample positive only in ITS in blood was also positive when its liquid culture was tested in real-time PCR. This condition occurred in other samples as well.
To test the detection limit of each PCR, tests with known Bartonella-DNA concentrations were performed as previously described [36]. Although one genome equivalent (GE) of B. henselae was amplified in at least one of several reactions performed in each PCR technique (analytical sensitivity), the detection limit of each test, which refers to the minimum GE that amplified in all reactions (diagnostic sensitivity), was 50 GE in conventional PCR, 20 GE in real-time PCR and 10 GE in nested PCR. These data allow us to hypothetically calculate the amount of GE required in the initial sample for detection by the methods described in this study, considering 1) the detection limit of each reaction, 2) the amount of initial sample used in each diagnosis stage (extraction: 1 mL of liquid culture and 200 μL of blood/PCR: 5 μL in conventional and real-time PCRs and 2.5 μL in nested PCR), and 3) the dilution effect (in case of liquid culture) ( Table 1). Sequencing Seventy-two amplicons presented sufficient quality for Sanger sequencing, including the 11 ITS amplicons. It was possible to sequence samples from 66 of the 102 positive donors. In six donors, more than one region could be sequenced (

Statistical analysis
The McNemar-Bowker test was performed to determine the best sample (blood or liquid culture) to be used regardless of the PCR technique. The result of 'detectable in any PCR' was considered the gold standard. The data show that the reactions conducted in liquid culture were significantly more effective in detecting DNA of Bartonella spp. than those performed in blood (p < 0.0001). Liquid culture was also better in relation to the negative predictive value and the negative odds ratio (Fig 2 and Table B in S1 Appendix). Bayesian LCM analysis was performed to determine the best PCR technique for blood or liquid culture samples and then to combine the results of blood and liquid culture, all of them using 'detectable in any PCR' as the gold standard (Fig 3). The results of conventional PCR for the ITS region were not used, as it was the only reaction targeting the Bartonella genus; all others targeted B. henselae.
Using the McNemar-Bowker test to analyze the results obtained only from whole blood samples, there was no difference in the concordance of results between nested PCR (ftsZ) and real-time PCR (gltA) (p value = 0.6547; McNemar-Bowker test, p > 0.05), demonstrating that they have similar diagnostic power in this type of sample ( Fig 3A and Tables C and D in S1 Appendix). This result was confirmed by the ROC curve method, which showed very close AUC values for these PCRs (Fig 4A).
No agreement was found between the PCR results from liquid culture according to the McNemar-Bowker test, suggesting that sensitivity is different between them (Fig 3B and Tables E and F in S1 Appendix). ROC curve method analyses showed that the AUC of real-time PCR was greater than the AUC of other PCR tests, i.e., real-time PCR had greater discriminatory diagnostic power for liquid culture samples (Fig 4B). Fig 3C shows the Bayesian LCM test combining liquid culture and blood sample PCR results, and together with the McNemar-Bowker test, it shows that the PCRs are different between them in this case (Tables G, H and I in S1 Appendix).
According to our literature review, few studies have been conducted with humans using several combined molecular tests and many samples. We found only ten studies published during the same period (from 2009 to 2022) about Bartonella sp. prevalence using PCR that analyzed at least 30 individuals, excluding blood donors [66][67][68][69][70][71][72][73][74] (Table K in S1 Appendix). Few of them have used PCR for different regions. Therefore, data found in the literature are not sufficient to determine the exact prevalence of bacteremia caused by Bartonella spp. [75].
A study conducted in Spain by Portillo et al. [72] analyzed samples from 97 sanitary workers using several techniques, such as serology for different species, direct blood extraction, liquid and solid cultures, and different molecular tests of these cultures, such as conventional and real-time PCR, with different primers for the ITS region, in addition to sequencing. With this combination of diagnostic tests, they obtained 83.1% positive results in IFA tests, and the DNA of Bartonella spp. was amplified by 21.6%. This percentage of molecular detection in asymptomatic individuals is very similar to our data.
The present study again analyzed samples from 500 donors previously studied with just one PCR from liquid culture. The results obtained now from whole blood and liquid culture using different PCR methodologies were compared and showed that detection was lower in reactions that used DNA directly extracted from whole blood (6% or 30/500) compared to liquid culture (15.4% or 77/500). In the McNemar-Bowker test, liquid culture showed better sensitivity and negative predictive value when compared to whole blood, which can be explained by the fact that a diagnosis obtained directly from blood is less effective than after enrichment culture [76]. The samples are from asymptomatic blood donors, so they must have low-level bacteremia, i.e., they are below the detection level of diagnostic sensitivity. After enrichment culture, bacterial multiplication may occur to a number above this detection limit. In immunocompetent humans, bacteremia caused by Bartonella spp. is estimated to be 1-10 GE/μL (i.e., 1,000-10,000 GE/mL) [15], which may lead to the real possibility of false negatives. Liquid culture of Bartonella spp. increases the sensitivity of detection of infection caused by these bacteria by molecular methods [76][77][78].
Twenty-five samples had DNA from Bartonella spp. detected from whole blood but were undetected when tested in liquid culture. This fact can be explained by the 'dilution effect' [79]. There was no increase in the number of bacteria in the culture, but dilution occurred in a large volume of culture medium, so the concentration of bacteria in the liquid culture was below the detection level, indicating amplification of nonviable bacteria. The fastidious characteristic of the bacterium combined with the presence of growth inhibitors (such as the use of antimicrobials, even if occasionally used as anti-inflammatories, as seen with sulfone, hydroxychloroquine, etc.) make this hypothesis even more probable [35]. Currently, several techniques must be used concomitantly to avoid false negative results [66].
As with other diagnostic methods, there is no consensus on the best primers and conditions for PCR to detect the DNA of Bartonella spp.. Several studies describe the 16S rRNA gene region, the 16S-23S rRNA intergenic locus (ITS), the citrate synthase gene, or the riboflavin synthase gene, the groEL gene, the ftsZ gene, the gltA gene, or the RNA polymerase beta subunit as the most efficient and promising for the detection and differentiation of the various species of Bartonella [80,81]. In addition to the primers that determine the region to be amplified and therefore the sensitivity of the reaction, the PCR technique also influences the success of the diagnosis. Nested PCR and real-time PCR can greatly increase detection sensitivity [78,[82][83][84]. In this study, the results obtained with statistical analysis show that conventional PCR in the gltA region is the least efficient technique to detect Bartonella sp.-DNA. Real-time PCR (gltA) is the best test for liquid culture, while for blood samples, no difference in sensitivity was found between nested (ftsZ) and real-time PCR (gltA) since confidence intervals for sensitivity overlap.
The great advantage of molecular diagnostic methods such as PCR is the fast result when compared to culture in addition to possible identification of the species causing the infection [85]. More sensitive and specific PCR tests allow quick diagnosis of the infection, even with low-level bacteremia. Maggi et al. developed, optimized, and validated droplet digital PCR (ddPCR), a new molecular technology, for the detection of Bartonella spp. DNA within several sample matrices. The ddPCR sensitivity (53/112) was significantly better than that of qPCR (6/ 112) when testing patient blood and enrichment blood culture samples [86]. Despite these advantages, PCR has some limitations: the possibility of false positive results due to contamination by control DNA or previously positive samples and false negative results for having less DNA than the detection limit. In addition, finding the pathogen DNA in the sample does not accurately indicate an active infection [79,87].
Several case reports [20,34,88-90] and a previous study with blood samples from cats [36] show that a combination of PCR and different samples increases the chances of detecting the pathogen. The results of this study agree with the literature and reinforce the need to combine several diagnostic tests to avoid false negatives.
In the previous project conducted with the same samples, the DNA of Bartonella spp. was detected in 16 donors [37]. Of these, the DNA was again amplified in only three samples, and only one that had been previously isolated by culture was detected again in real-time PCR. (Table L in S1 Appendix). This divergence in the results from the two studies developed with the samples of 500 blood donors shows how challenging the laboratory diagnosis of Bartonella spp. infection can be, since the samples from which isolates were previously obtained were supposed to have positive reactions, which did not happen in most samples. In the first study with these samples, only one conventional PCR genus-specific reaction was performed for the ITS region using DNA extracted from the liquid culture. Several factors can explain this divergence, including low-level bacteremia (1-10 GE/μL), especially for asymptomatic individuals. Therefore, there must be a small amount of pathogen DNA close to the detection limit. The aliquot used in the reaction may not have the amount required for amplification (Fig A in  S1 Appendix).
Additionally, a stochastic (random) variation of the PCR amplification process occurs in the analysis of low amounts of DNA. Stochastic effects are seen as a fluctuation of results between replicated analyses [79]. For this reason, even a combination of several techniques does not prevent false negative results. In a previous study that used the same samples, the DNA of Bartonella spp. was detected in only 3.2% (16/500) of liquid culture samples using conventional PCR, and six of them were isolated in solid culture. In five of these six isolates, we were unable to detect Bartonella sp.-DNA [37].
Edouard et al. argue that to confirm a diagnosis of bartonelloses using exclusively the PCR technique, only samples with the DNA of Bartonella spp. detected in at least two different genome regions [68] should be considered. In this case, the possibility of false positives is reduced, and consequently, the specificity increases, but sensitivity is lost. Even considering this criterion, 35 (7%) samples were positive in our study with reactions in two different genome regions. If the six samples from which isolates were obtained in the previous study were added, we obtained 41 B. henselae DNA-detected samples (8.2%). None of the samples that originated these six isolates met the criterion of two distinct detected regions even in this current study using different PCRs.
As bartonelloses are caused by fastidious bacteria and low-level bacteremia is characteristic of the infection, it would not be advisable to use the criterion of two different gene region detections to confirm the diagnosis. Then, when adding up all the samples with Bartonella sp.-DNA detection in the two studies, 115/500 donors had Bartonella spp. detected, which corresponds to 23%. This result is close to the percentage of bloodstream infection in sanitary workers found in the study by Portillo et al. [72].
Cases reported in the literature [34, 91,92] show that the low sensitivity of molecular tests may impact clinical practice. The five most common manifestations related to Bartonella spp. (CSD, bacillary angiomatosis, bacillary peliosis, culture-negative endocarditis and fever of undetermined origin) [93,94] are unquestionable and justify more investments in studies of this kind.
The laboratory diagnosis of Bartonella spp. is a challenge for several reasons: first, the fastidious characteristic of the bacterium, which makes laboratory culture an obstacle; second, the fact that it causes cyclic and low bacteremia; and finally, the lack of specific and sensitive tests for its diagnosis [15,95]. Combining methods is required to reduce false negatives. Further efforts should be dedicated to improving the diagnostic methods and ensuring better sensitivity to screen for infection by Bartonella spp..
The statistical analysis using all results of blood and liquid culture samples showed that, regardless of the sample, the sensitivity differs with the PCR types (conventional, nested and real-time PCR) and targets of gene regions.
The use of three different PCR tests with two types of samples (blood and liquid culture) increased the possibility of detecting Bartonella spp. considering that, in a previous project, 3.2% of blood donors were positive, and in this project, this rate increased to 20.4%. However, the combination of techniques did not prevent false-negative results since 13 donors who were positive in the previous project were not positive again.

Conclusions
More than one-fifth of blood donors had at least one B. henselae DNA detected by a PCR test among the eight molecular reactions performed. Seven percent had the DNA detected for two or more distinct regions.
The statistical analysis using all results of blood and liquid culture samples showed that, regardless of the sample, the sensitivity differs with the PCR types (conventional, nested and real-time PCR) and targets of gene regions.
The results of our study indicate that public health authorities must review the risks and the impact of the transmission of Bartonella spp. through blood transfusions, especially for immunocompromised patients. Low-level bacteremia and the fastidious characteristics of the bacterium are challenges to laboratory diagnosis.
Supporting information S1 Appendix.